The protein phosphatase 2C (PP2C) family represents one of the four major protein Ser/Thr phosphatase activities in mammalian cells and contains at least 13 distinct gene products. Although PP2C family members regulate a variety of cellular functions, mechanisms of regulation of their activities are largely unknown. Here, we show that PP2Cζ, a PP2C family member that is enriched in testicular germ cells, is phosphorylated by c-Jun NH2-terminal kinase (JNK) but not by p38 in vitro. Mass spectrometry and mutational analyses demonstrated that phosphorylation occurs at Ser92, Thr202, and Thr205 of PP2Cζ. Phosphorylation of these Ser and Thr residues of PP2Cζ ectopically expressed in 293 cells was enhanced by osmotic stress and was attenuated by a JNK inhibitor but not by p38 or MEK inhibitors. Phosphorylation of PP2Cζ by TAK1-activated JNK repressed its phosphatase activity in cells, and alanine mutation at Ser92 but not at Thr202 or Thr205 suppressed this inhibition. Taken together, these results suggest that specific phosphorylation of PP2Cζ at Ser92 by stress-activated JNK attenuates its phosphatase activity in cells.